Comparison of luteal support protocols in fresh IVF/ICSI cycles: a network meta-analysis

Despite the proven superiority of various luteal phase support protocols (LPS) over placebo in view of improved pregnancy rates in fresh cycles of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) cycles, there is ongoing controversy over specific LPS protocol selection, dosage, and duration. The aim of the present study was to identify the optimal LPS under six core aspects of ART success, clinical pregnancy, live birth as primary outcomes and biochemical pregnancy, miscarriage, multiple pregnancy, ovarian hyperstimulation syndrome (OHSS) events as secondary outcomes. Twelve databases, namely Embase (OVID), MEDLINE (R) (OVID), GlobalHealth (Archive), GlobalHealth, Health and Psychosocial Instruments, Maternity & Infant Care Database (MIDIRS), APA PsycTests, ClinicalTrials.gov, HMIC Health Management Information Consortium, CENTRAL, Web of Science, Scopus and two prospective registers, MedRxiv, Research Square were searched from inception to Aug.1st, 2023, (PROSPERO Registration: CRD42022358986). Only Randomised Controlled Trials (RCTs) were included. Bayesian network meta-analysis (NMA) model was employed for outcome analysis, presenting fixed effects, odds ratios (ORs) with 95% credibility intervals (CrIs). Vaginal Progesterone (VP) was considered the reference LPS given its’ clinical relevance. Seventy-six RCTs, comparing 22 interventions, and including 26,536 participants were included in the present NMA. Overall CiNeMa risk of bias was deemed moderate, and network inconsistency per outcome was deemed low (Multiple pregnancy χ2: 0.11, OHSS χ2: 0.26), moderate (Clinical Pregnancy: χ2: 7.02, Live birth χ2: 10.95, Biochemical pregnancy: χ2: 6.60, Miscarriage: χ2: 11.305). Combinatorial regimens, with subcutaneous GnRH-a (SCGnRH-a) on a vaginal progesterone base and oral oestrogen (OE) appeared to overall improve clinical pregnancy events; VP + OE + SCGnRH-a [OR 1.57 (95% CrI 1.11 to 2.22)], VP + SCGnRH-a [OR 1.28 (95% CrI 1.05 to 1.55)] as well as live pregnancy events, VP + OE + SCGnRH-a [OR 8.81 (95% CrI 2.35 to 39.1)], VP + SCGnRH-a [OR 1.76 (95% CrI 1.45 to 2.15)]. Equally, the progesterone free LPS, intramuscular human chorionic gonadotrophin, [OR 9.67 (95% CrI 2.34, 73.2)] was also found to increase live birth events, however was also associated with an increased probability of ovarian hyperstimulation, [OR 1.64 (95% CrI 0.75, 3.71)]. The combination of intramuscular and vaginal progesterone was associated with higher multiple pregnancy events, [OR 7.09 (95% CrI 2.49, 31.)]. Of all LPS protocols, VP + SC GnRH-a was found to significantly reduce miscarriage events, OR 0.54 (95% CrI 0.37 to 0.80). Subgroup analysis according to ovarian stimulation (OS) protocol revealed that the optimal LPS across both long and short OS, taking into account increase in live birth and reduction in miscarriage as well as OHSS events, was VP + SCGnRH-a, with an OR 2.89 [95% CrI 1.08, 2.96] and OR 2.84 [95% CrI 1.35, 6.26] respectively. Overall, NMA data suggest that combinatorial treatments, with the addition of SCGnRH-a on a VP base result in improved clinical pregnancy and live birth events in both GnRH-agonist and antagonist ovarian stimulation protocols.


Outcomes
The NMA primary outcomes were clinical pregnancy, defined as the presence of a gestational sac, with or without a fetal heartbeat on ultrasonography (US) and live birth, defined as the number of deliveries that resulted in live born neonate/s.Regarding live birth, singleton and non-singleton deliveries were considered as a single event.Secondary outcomes included biochemical pregnancy, defined as positive hCG test but without US verification 2 weeks following embryo transfer (ET), miscarriage defined as the spontaneous loss of a pregnancy before the 20th week, multiple pregnancy was defined as non-singleton clinical pregnancy and OHSS events.Crude events were collected per included study, and therefore no homogenisation of extracted data was required.

Data analysis
Effect estimates were calculated as odds ratios (ORs) for all outcomes, given that all were dichotomous, with respective 95% credibility intervals (95% CrIs) using Bayesian network and pair-wise meta-analysis 14 (Fig. 5, Fig. S2-S10).Of note, a credibility interval is an interval within which an unobserved parameter value falls with a particular probability in Bayesian statistics comparable to the 95% Confidence interval commonly seen in frequentist statistics 15 .Network meta-analysis iterations were conducted with MetaInsight visual R package 16 .NMA was conducted using a fixed-effects model within a Bayesian setting, as unequal heterogeneity across all comparisons was assumed.Vaginal Progesterone (VP) was used as the reference treatment given its proven superiority over placebo and the NICE guideline recommendations 17 .A hierarchy of treatments was calculated for each outcome, based on the p-scores and SUCRA ratings.Summary of the rank distribution of LPS treatments, interpreted as the estimated proportion of treatments worse than the treatment of reference (VP) was displayed by Litmus Rank-O-Gram graphs and Radial SUCRA 18 (Fig. S5-S6).Transitivity assumption was evaluated by comparing the distribution of key study characteristics across studies grouped by comparison (age and BMI).We assessed inconsistency between direct and indirect sources of evidence using global and local approaches.We assessed global inconsistency by using a design-by-treatment test 19,20 .Local inconsistency was evaluated by using the back calculation and separate indirect from direct design evidence methods, comparing direct and indirect evidence for each pairwise treatment comparison and node-splitting model 21 (Table S2-S3; Fig. S3-S4).Possible heterogeneity of treatment effects and the robustness of findings was explored by subgroup network meta-analyses including only trials at overall low and medium risk of bias (  subgroup analysis was conducted on trials using either standard (long) GnRH agonist or standard (short) GnRH antagonist protocol for ovarian stimulation to limit data heterogeneity.If mixed populations were included in the original publication, a cut-off of ≥ 65% of patients being treated with either of the protocols, was employed to categorise studies according to subgroup (Table S4).Mixmeta package in R v4.1.2was employed for confounder exploration in a network meta-regression model.Gelman network convergence, network deviance and ranking analysis were conducted to quantify overall network discordance (Fig. S9-S10).Intergroup differences regarding demographic and treatment parameters were quantified, where appropriate by ANOVA (for parametric distributed variables e.g., Age, BMI) or Kruskal-Wallis test (non-parametric distribution of variables, e.g., all remaining variables).Multilevel network meta-regression for the embryological parameters (number of transferred embryos, number of retrieved and mature oocytes, peak estradiol, % of day 3 embryos transferred) was undertaken for both primary and secondary outcomes 22 (Table S11-S12).

Risk of bias assessment
Within-study bias was assessed with the Cochrane risk of bias tool RoB2 23 and the certainty of evidence using the GRADE Framework (Table 2).Overall network risk of bias was assessed with the Network Meta-Analysis framework (CINeMA) 24 (Table S5-S10).Small-study effects and publication bias for each treatment pair was assessed using a contour-enhanced funnel plot.2B,C).Duration of infertility was of a median of 4.96 years [IQR 3.98, 6.10] (Fig. 3A).The population percentage diagnosed with primary infertility was 29.6% [Range: 10.9 to 42.62%] and secondary infertility was 34.5% [Range: 16.1 to 84.65%] and were not found to significantly differ across comparator groups (Fig. 3A-C).Median values of basal AMH, LH, FSH, progesterone levels on HCG trigger, progesterone levels on embryo transfer (ET) day, and endometrial thickness on ET day, per LPS were not found to be significantly different in comparison to the VP group (Figs.3D-F, 4A-C).Regarding OS protocol, 54.69% of the participants underwent ovarian stimulation with a standard (long) GnRH agonist while 18.17% with a standard (short) GnRH antagonist protocol.A 1.05% underwent OS via clomiphene and HMG, 0.96% via a microdose flare and 0.54% by an ultrashort GnRH protocol (Fig. 4D) 101 .The remaining 24.59% of the participants underwent either a standard long or short OS protocol however the distribution was not noted in the original studies.Characteristics of embryo transfers were not consistently reported across arms of included studies (Table S.11).Of note, 20 of the 76 studies, failed to report upon these variables.

Participant and treatment characteristics
Regarding LPS protocols, schemas were segregated by active compound to explore variations of dosage (median dosage and maximum dosage), initiation day, duration of LPS (weeks) as well route of administration (Tables 1, 3).The majority of LPS protocols were initiated on the oocyte pickup day (OPU), and duration of 8 weeks (SD = 2).No significant differences were noted amongst LPS protocols regarding implantation 24.55% [IQR 18.17, 28.9] or fertilisation 63.6% [IQR 61, 78.9] median rates (Fig. S1).

Data synthesis and network meta-analysis
VP was considered as the reference treatment as previously mentioned (NICE guidelines 17 .In NMA, effect size estimates suggested that all LPS protocols were consistently superior to placebo, employed as a negative control for both primary and secondary outcomes, regardless of risk of bias sensitivity analysis (Fig. 5, Fig. S2-S10, Tables S2-S4).
Subgroup analysis of low and medium risk of bias studies (Figs.S7, S8, S10, Table 2) and node-splitting (Table S2-S3) did not significantly alter cumulative effects analysis or residual deviance (Fig. S10A-F).Optimal LPS per OS, long (Gonadotropin releasing hormone agonist) vs. short (GnRH antagonist) protocol, was explored to identify further sources of heterogeneity and to delineate whether a particular LPS appears to yield improved clinical outcomes in association with specific ovarian stimulation protocols (Table 4, Table S4).In view of live birth events, the following protocols were deemed optimal for participants that underwent OS by standard GnRH agonist protocol: Of the aforementioned, the VP + OE, VP + SCGNRH-a and IMHCG comparators had a "High" GRADE rating while the VP + OE + SCGNRH-a protocol was also associated with a higher probability of miscarriage when used in combination with a GnRH agonist OS protocol, [OR 3.93 (1.69, 10.1)].On the contrary, optimal luteal support protocols for standard GnRH antagonist OS were (a) IMHCG [OR 3.2 (95% CrI 1.54, 334.), "low" GRADE] and (b) VP + SCGNRH [OR 2.84 (95% CrI 1.35, 6.24), "High" GRADE] presenting the optimal LPS options across short protocols.Of note, IMHCG was also associated with a higher probability of miscarriage when used in conjunction with a short OS protocol [OR 2.11 (95% CrI 0.75, 6.40), high GRADE] while the opposite held true for VP + SCGNRH, which was associated with lower probability of miscarriage in short OS [OR 0.54 (95% CrI 0.37, 0.80), high GRADE].Network meta-regression for all outcomes, according to embryological parameters, did not significantly alter effect sizes (Table S11-S12).
Overall, NMA data suggest that combinatorial treatments, with the addition of SCGNRH-a on a VP base results in improved clinical pregnancy and live birth events and reduced miscarriage events in participants undergoing OS either a standard GnRH antagonist or agonist protocol.However, participants undergoing a long GnRH protocol OS appear to benefit more from IMHCG as LPS while participants undergoing a short GnRH protocol OS appear to benefit more from VP + SCGNRH, considering the reduction of miscarriage events of these luteal support protocols in conjunction to OS.

Discussion
This study is based on 76 RCTs, including 26,536 participants randomly assigned to 22 LPS protocols including non-exposure.Given the plethora of previous data suggesting that any LPS protocol is superior to non-exposure, the most widely employed LPS, vaginal progesterone, was set as a reference treatment 3,17 .Overall, meta-synthesized data presented here, suggest that combinatorial treatments, those with the addition of SCGnRH on a VP base result in improved clinical pregnancy, OR 1.28 (95% CrI 1.05 to 1.55) and live birth events, OR 1.76 (95% CrI 1.45 to 2.15) with high confidence in evidence.Of note, addition of oral estradiol to a VP + SCGNRH-a LPS, resulted in further improvement of clinical pregnancy events by 29% and 44% increase of a clinical pregnancy and live birth odds respectively.Of note, participants undergoing a long GnRH protocol OS appeared to benefit more from progesterone free LPS such as IMHCG in view of increased live birth, OR 1.57 (95% CrI 2.24 to 71.9) and reduced miscarriage events, OR 1.57 (95% CrI 2.24 to 71.9).However, participants appeared to be at a higher risk of OHSS, OR 1.64 (95% CrI 0.74 to 3.73).On the other hand, participants undergoing a short GnRH OS protocol appeared to benefit more from VP + SCGNRH with a live birth OR 2.84 (95% CrI 1.35 to 6.26), however while the probability of miscarriage was significantly reduced, OR 0.55 (95% CrI 0.38 to 0.80), the probability of multiple pregnancy significantly increased, OR 8.34 (95% CrI 2.57 to 37.6).
Luteal support is a critical aspect of IVF/ICSI cycles as it aids in maintaining the endometrial lining, in turn promoting embryo implantation, and supporting early pregnancy.In fresh IVF cycles, luteal support management can pose several challenges, including timing and duration of administration, individual outcome variability and tolerability of LPSs that may impact upon the success rates of the cycle.The effectiveness of luteal support in achieving live birth and clinical pregnancy rates is dependent on the timing of its administration [104][105][106] .Various studies have examined the optimal timepoint to initiate LPS, with only two out of five RCTs reporting statistically significant results 104 .Earlier evidence had suggested that delayed administration of LPS [(24 h after ovum pick-up (OPU)] may be more advantageous than pre-OPU administration (12 h prior to OPU) 106 .Williams et al. found initiating LPS on day 3 post OPU to be significantly better than delaying it until day 6 105 .Overall, these studies suggest that the optimal time for LPS administration is from the evening of OPU up until 3 days post OPU.Present NMA evidence suggested that the majority of studies favoured LPS initiation on the day of OPU (within the 24 h timeframe following the procedure), including for LPS protocols generating superior results namely, VP + SCGNRH-a and VP + OE + SCGNRH-a.Equally important to the LPS initiation timing, is the duration of luteal support administration.A recent meta-analysis including 1297 participants, indicating that continuing progesterone for two weeks after a positive pregnancy test did not have any significant impact on miscarriage or delivery rates 106 .The same study suggested that it is unnecessary to continue LPS for up to 10 weeks of pregnancy with further studies reaching to the same conclusion [107][108][109] .However, ESHRE 2020 recommendations suggest  12] coinciding with ultrasonographic evidence of fetal motion and the concept of the luteo-placental shift 110,111 .
In addition, while initiation and duration of LPS treatment may appear more standardised, the selection of optimal type and dose of luteal support is largely individualised and dependent upon participant factors such as age, BMI, and reproductive history.Regardless of clinical and demographic parameters, undoubedtly the most important parameter affecting LPS selection and duration of perscription, is fundamentaly influenced by patient preference, which is in turn heavily reliant upon LPS side effect profile and tolerability, patient compliance and cost.For example, in view of treatment acceptability, IM progesterone has been widely available prior to vaginal formulation becoming available, and has been shown to have superior absorption and achieve stable serum concentration shortly after administration 109,112,113 .Nonetheless, administration complications involving pain, higher risk of infection, sterile abscess formation, and even rarely eosinophilic pneumonia as well as practical impediments requiring daily visits and injections, have necessitated the exploration of alternative more convinient routes, such as the one offered by the vaginal preparation 114 .Currently, vaginal progesterone products are administered in various ways, including pessaries, capsules, tablets, gel, and inserts which can achieve maximum serum concentration of progesterone after 3-8 h of administration, and by daily doses of 300-600 mg may achieve adequate available plasma levels 115 .Evidence has also shown that a 300-600 mg of vaginal micronized progesterone daily can induce similar endometrial maturation as 100 mg intramuscular progesterone daily 109 .By enabling direct transport of "first uterine pass" progesterone from the vagina to the uterus, vaginal preparations achieve adequate tissue levels of progesterone with lower circulating levels, indicating acceptable bioavailability 111 .
Given the improved outcomes regarding clinical pregnancy and live birth, achieved by VP + SCGNRH-a and VP + OE + SCGnRH-a combinatorial treatments, shown in the present work, a mention to route and dosage of gonadotropin-releasing hormone agonist is warranted.The use of GnRH-a for LPS was suggested following accidental use of GnRH agonist during this phase which resulted in improved implantation rates 116 .The effect of GnRH agonist has been observed at three levels: support of the corpus luteum through pituitary LH secretion, direct effects on the embryo and implantation process, and the effect upon trophectoderm cells and endometrial GnRH receptors 77,116,117 .A meta-analysis showed that administering a single dose of GnRH-a increased the implantation rate in cycles with GnRH antagonist and long GnRH-a protocols, clinical pregnancy rate per transfer, and ongoing pregnancy rate 118 , whilst another revealed that the use of GnRH-a for LPS significantly improved live birth rate, clinical pregnancy rate, and ongoing pregnancy rate 119 .An additional study demonstrated that a single dose of GnRH-a had similar efficacy as three doses of hCG 120 .One can hypothesise that the addition of a GnRH agonist can bimodally support the corpus luteum by stimulating the release of gonadotrophins from the pituitary gland, and by directly influencing the endometrium through interaction with GnRH receptors.Furthermore, research suggests that administering a single dose of GnRH agonist during the luteal phase enhances rates of pregnancy, implantation, delivery, and birth among recipients of donated oocytes whose ovulation was suppressed and corpus luteum was absent, suggesting a potential direct impact of GnRH agonist on the embryo 77,98,116,119 .The present work has highlighted that a single SCGNRH administration in addition to a VP protocol, can positively impact on IVF/ICSI outcomes especially in patients undergoing GnRH antagonist OS and could be reserved for more challenging cycles to optimise results.Conversely, in view of the improved clinical pregnancy and live birth outcomes achieved by the addition of oral estradiol in the VP + SCGNRH-a protocol, exploration of the possible synergistic effects of this compound is necessitated.However, a Cochrane meta-analysis did not find evidence to support routinely administering estrogen with progesterone in IVF cycles 9 .In antagonist cycles, progesterone levels surge, leading to a rebound decrease in serum estradiol, which in turn has formulated the hypothesis that adding doses of 2-6 mg/day of estradiol could be beneficial 111 .However, contemporary systematic reviews failed to confirm the beneficial effects of oral or any route of estradiol addition to progesterone LPS upon pregnancy outcomes [120][121][122] .Of note, novel LPS regimens involving intranasal GnRH administration has been shown promising results regarding clinical pregnancy rates and treatment tolerability however given the scarse RCT evidence, further, adequately powered, RCTs would be required to allow recommendations regarding this LPS regimen 117,123 .
In addition, the present work has shown that progesterone free LPS protocols, such as intramuscular hCG, may be equal, if not more effective that progesterone-based LPS in view of live birth outcomes, especially in patients undergoing a GnRH agonist OS protocol.HCG, by mimicking LH pulsatility, was initially considered the primary choice for LPS as it stimulates the corpus luteum to produce progesterone continuously.However, this approach has drawbacks, as it can elevate the risk of OHSS, a hypothesis which was also confirmed by the present NMA, albeit lacking statistical significance, OR 1.64 [95% CrI 0.75, 3.71].

Limitations and future perspectives
The optimal protocol for luteal support is a constantly evolving field of research in artificial reproduction.In view of the plethora of available LPS protocols, NMA precision of estimates provides a more comprehensive understanding of the comparative effectiveness of different protocols.In the present work, only RCT data have been employed to reach meaningful conclusions limiting inherent bias of diverse participant populations, with add-on sensitivity analysis targeted at low and moderate risk of bias studies and ovarian stimulation protocol to further explore confounding factors and detect sources of heterogeneity.Given the anticipated diversity of measured outcomes, a bayesian meta-synthesis approach has been adopted to account for the expected heterogeneity and to incorporate modelling flexibility by allowing for posterior distributions interpreted as SUCRA probabilities with the later enabling crisper communication of the uncertainty in the treatment effects estimates.
On this note, in the present study, LPS protocols have been treated as unique comparator entities, allowing for assessment of selected outcomes on a protocol-rather than a compound-level.However, side effect and safety profile of combinatorial treatments has not been assessed and therefore a significant confounder in tolerability and in turn, compliance, especially in the context of combinatorial LPS, remains to be investigated.Additionally, cost-effectiveness analysis has not been undertaken, which needs to be factored in a joined patient and clinical decision-making.Moreover, while reported, the present study did not aim to clarify of optimal initiation/cessation timing of LPS or the optimal dosage and therefore to produce concrete recommendations regarding these LPS parameters, further studies with relevant designs should be implemented.Notably, included studies were significantly heterogeneous in terms of reporting the characteristics of embryo transfers.Abeit no statistical difference was reported for variables such as follicles retrieved, peak oestradiol levels and number of embryos transferred in individuals studies, confounding effects cannot be confidently excluded.Lastly, OHSS events were found to be considerably under-reported across RCTs with only 15 studies noting such events.Reflecting on the implications of OHSS upon both the patient clinical management as well as the success of the IVF/ICSI, it would be strongly recommended that future RCTs would thoroughly record OHSS events across study arms.Overall, luteal support management in fresh IVF cycles is a complex and dynamic process that calls for careful consideration and individualised LPS selection to achieve optimal outcomes.

Conclusion
Herein meta-synthesized data suggest that combinatorial treatments, with the addition of subcutaneous GnRH agonist, on a vaginally administered progesterone LPS base, results in improved clinical pregnancy and live birth events.However, the side-effect and tolerability profile of such combinatorial LPS protocols needs to be thoroughly investigated prior to their wide-scale adoption in clinical practice.

Figure 1 .
Figure 1.PRISMA Flow chart.PRISMA 2020 flow diagram for new systematic reviews which included searches of databases, registers and other sources.